Soluble biological substances attached to carriers have many uses in diagnostic tests, enzyme processes, and affinity purifications. For example, attachment of antibodies or antigens to a carrier allows their immunological partners to be easily removed from a mixture of many substances. Similarly, attaching enzymes to a carrier allows them to be easily removed from a reaction mixture or to be used in a continuous flow process. Heterogeneous radioimmunoassays and enzyme immunoassays rely on attachment of one or more of the reactants to a solid phase to enable separation from the free reactants. Agglutination assays (to determine the presence of an antigen or an antibody in a fluid) utilize indicator or carrier particles (on which are carried the appropriate immunological material) in order to make the immunological complex more easily visible. Separation and identification of cells, cellular constituents, and bacteria are aided by antibodies or antigens coupled to solids. Biological particles will, for example, specifically adhere to solids coated with appropriate antibodies and antigens so that separation from other particles can be affected. Identification of biological particles can be made through the specific adherence of small particles coated with appropriate antibody or antigen. These small particles can incorporate a substance such as a fluorescent dye, radioactive tracer, or electron dense substance which make their presence more readily detectable.
The currently available simple procedures for bioactive material testing, for field test methods (e.g., over the counter pregnancy test kits, rely primarily on agglutination reactions or on enzyme catalyzed color reactions. These procedures use one of several core materials in their reaction, i.e., they use sheep's red blood cells, latex particles, or killed Staphylococcus cells as carriers. Such procedures are quite fast, usually taking less than an hour to complete. However, such procedures have several significant drawbacks. First, the biological core materials, when utilized, have production difficulties that can lead to non-reproducible test results for specific bioactive materials. Second, the maximum sensitivity level for reproducible, reliable results, is in the microgram per milliliter concentration range. This is far above the 1-10 nanogram per milliliter sensitivity which is required for early hormonal, viral or bacteriological analyte detection.
The agglutination procedures often require the careful manipulation of two solutions so that a successful reaction will occur to cause agglutination. Manipulations usually take the form of stirring small volumes of the solution on a specially designed flat surface and then waiting for the agglutinates to appear, or mixing two solutions by rocking a specially designed flat surface back and forth steadily until agglutinates appear. In another form of common agglutination test two regents are mixed in a test tube. As the agglutinate forms, it becomes insoluble and precipitates out to form a pattern on the tube bottom. This is observed and is accorded a negative or positive classification by its shape. The appearance of the agglutination reaction is in no way standardized and is therefore easily misinterpreted by persons with inadequate training or instruction. The agglutination procedures are very technique dependent. Generally it requires about two hours of training to qualify an operator already familiar with laboratory techniques to carry out the slide-type test. When the agglutination procedure is carried out in a test tube as described above, it is extremely sensitive to vibration, temperature changes and mixing techniques.
The enzyme catalyzed reaction of the prior art require preparation of biological substrates for the enzymatic reaction and careful manipulation of several reagent substances to first start the reaction and then, after a rather precise time interval, to stop the reaction before an observation can be completed. The enzyme test procedures are sensitive to temperature changes. During the reaction incubation the ambient temperature must be assumed to be about 22.degree. C. The reactions are very difficult to stop, sometimes requiring the use of strong bases, e.g., 10 N sodium hydroxide for this purpose. And, they are quite time dependent. That is, they are kinetic reactions that must be closely timed for accurate and precise test results.
The prior art field tests, or home use tests, for bioactive materials thus have a number of very serious problems.